We previously reported that the selection of wild-type HIV-1 strains in the presence of each of 8 FDA-approved protease inhibitors (PIs) readily gave HIV-1 variants resistant to each PI over 20 to 67 weeks. However, when HIV-1 was selected against DRV using standardized selection protocols, the development of HIV-1 variants resistant to DRV was not seen or much delayed, and no significant DRV-resistance-associated amino acid substitutions were identified. Indeed, an infectious HIV-1NL4-3 clone (rHIVWT) failed to replicate in the presence of over 0.075 uM DRV even after 50 weeks of selection. As previously reported, the highly DRV-resistant HIV-1 variant, HIVDRVRP51, contains four major DRV-resistance-associated amino acid substitutions (V32I, L33F, I54M, and I84V) in its protease. However, the role of each of the four substitutions in the development of HIV-1's DRV-resistance has remained to be determined. Thus, we newly generated a variety of recombinant infectious HIV-1 clones. When three such recombinant clones (rHIVV32I/L33F/I54M/I84V, rHIVV32I/I54M, and rHIVL33F/I84V) were propagated in the increasing concentrations of DRV, rHIVV32I/L33F/I54M/I84V readily acquired high-level DRV-resistance and replicated in the presence of 5 uM by 17 weeks of selection, followed by rHIVV32I/I54M and rHIVL33F/I84V, which vigorously replicated in the presence of 1 uM by the end of 22 and 26 weeks of selection, respectively. rHIVV32I, rHIVI54M, and rHIVI84V acquired DRV-resistance relatively quickly and replicated in the presence of over 1 uM DRV by the end of 36 weeks of selection, while rHIVL33F failed to acquire DRV-resistance by the end of week 50 of selection. The amino acid sequence of the protease-encoding region of each variant was directly determined using proviral DNA isolated from the HIV-1-producing MT-4 cells at various time points of the selection. HIVWT examined at week 50 of selection (HIVWT-WK50) had acquired three substitutions, M46L, K55N, and V82I, by 50 weeks. HIVL33F-WK50, which apparently did not acquire DRV-resistance, had acquired only K43T. It was of note that all the six clones that eventually developed DRV-resistance (HIVV32I/L33F/I54M/I84V-WK17, HIVV32I/I54M-WK22 HIVL33F/I84V-WK26, HIVI84V-WK29, HIVV32I-WK36, and HIVI54M-WK36) had contained V32I substitution, although other substitutions such as L10F, L33F, M46I, A71V, and I84V had been acquired in parts of the 6 clones, suggesting that V32I substitution might have played an important role in the pathway of DRV-resistance development. It was also noted that in 5 of the 6 clones, A71V had emerged in quite early stages of DRV selection (by the end of 5-8 weeks of selection), followed by the emergence of V32I substitution. It is possible that the presence of A71V might predispose to HIV-1's acquisition of V32I. We next attempted to determine how each of the four amino acid substitutions contributed to the high-level DRV-resistance of HIVDRVRP51. To this end, we first replaced the gag- and protease-encoding genes of rHIVWT with those derived from HIVDRVRP51 and obtained recombinant infectious HIV-1 clone, designated as rHIVDRVRP51. This rHIVDRVRP51 was confirmed to be highly resistant to DRV showing an IC50 value of 330 nM, 106-fold greater than that of rHIVWT. We then reverted each of the four substitutions to the wild-type amino acid in rHIVDRVRP51. A newly generated infectious clone, rHIVDRVRP51I32V, in which the substitution V32I was reverted back to Val, was found to be only moderately resistant to DRV with an IC50 value of 29 nM. The IC50 values of rHIVDRVRP51F33L, rHIVDRVRP51M54I, and rHIVDRVRP51V84I turned out to be 120, 43, and 28 nM. These data strongly suggested that the order of the magnitude of contribution to the high-level DRV-resistance was: V32I=I84VI54ML33F. We subsequently introduced all the four substitutions or each single substitution into HIVNL4-3 (rHIVWT), generating rHIVV32I/L33F/I54M/I84V, rHIVV32I, rHIVL33F, rHIVI54M, and rHIVI84V. As expected, rHIVV32I/L33F/I54M/I84V proved to be highly resistant to DRV with an IC50 of 639 nM, while rHIVL33F, rHIVI54M, and rHIVI84V were as sensitive as rHIVWT with IC50 values of 3 nM, virtually identical to that of rHIVWT. Of note, rHIVV32I was hypersensitive to DRV with an IC50 value of 0.2 nM, 16.7-fold more sensitive to DRV as compared to rHIVWT, suggesting that since the emergence of DRV-hypersensitive HIVV32I would be prohibitive in the presence of DRV, rHIVWT is not likely to directly acquire V32I substitution. We next generated various recombinant infectious HIV-1 clones and determined their susceptibility to DRV. The addition of A71V to rHIVV32I that was hypersensitive to DRV (IC50=0.2 nM), generating rHIVV32I/A71V, deprived the rHIVV32I of the hypersensitivity to DRV and the IC50 of rHIVV32I/A71V became virtually the same as that of rHIVWT (2.8 versus 3.1 nM). Two clones, rHIVV32I/I54M and rHIVV32I/I84V, were also found hypersensitive to DRV with IC50s of 1.7 and 0.42 nM, suggesting that these two clones would not emerge in the presence of DRV. However, the addition of A71V to these two clones, generating rHIVV32I/I54M/A71V and rHIVV32I/A71V/I84V, acquired significant resistance to DRV with their IC50 values of 27 and 35 nM, respectively. These data suggest that the presence of A71V predisposes HIV-1 to its acquisition of V32I. We then analyzed the interactions of V32I mutant protease with DRV. The bis-THF has an enhanced interaction with mutant Ile32 than with wild-type Val32. Ile32' also has enhanced interactions with the aminobenzene group of DRV than does Val32 in the wild-type protease. Overall, the Connolly surface interactions suggest better interactions of DRV with both Ile32 and Ile32' of mutant protease than with Val32 and Val32' of wild-type protease. In conclusion, we demonstrated that one of the four critical amino acid substitutions, V32I, serves as a key substitution, which hardly occurs in in vitro selection attempts, but once it occurs, predisposes HIV-1 to develop high-level DRV resistance. The present data not only well explain the mechanisms of DRV's high genetic barrier but also alarm that the initiation/continuation of DRV-containing regimens in individuals harboring HIV-1 variants with V32I substitution must be carefully considered/monitored. We have also demonstrated multiple novel protease inhibitors including (i) protease inhibitors containing C-5-modified bis-THF and aminobenzothiazoleas P2 or P2' ligands; (ii) central nervous system (CNS)-targeting protease inhibitors; (iii) Halogen-containing HIV-1 protease inhibitors (GRL-003-15 and GRL-001-15); (iv) protease inhibitors containing P2 bicyclic oxazolidinone scaffold; and (v) Protease Inhibitors Containing P2 Tricyclic Fused Ring Systems.